Synthetic Fab Antibody Library Screening Service

Introduction of Synthetic Fab Antibody Library Screening ServiceFab

Synthetic antibody library refers to antibody variable region sequences artificially designed and synthesized based on structure and function. In contrast, the skeleton region sequences are fixedly designed and synthesized according to the information in the human antibody library.

The advantages of a synthetic antibody library are as follows: (1) The randomness of the library increases and the capacity of the library is large; (2) Some difficult antibodies can be screened without immunizing animals; (3) Compared with the naïve antibody library, the design of the synthetic antibody library is diverse.

Disadvantages of synthetic antibody library: artificially designed sequence diversity, no evolution in vivo, prone to abnormal protein modification or amino acid clusters, low expression level, and easy degradation, so the screened antibodies need to undergo necessary antibody optimization to be more drugable.

Alpha Lifetech Can Provide

Alpha Lifetech has been deeply engaged in antibody phage display technology for many years. It has built a perfect stable Phage display technology platform that saves time for scientific research time or project research and facilitates subsequent production. Alpha Lifetech can also provide customers with a Phage display selection the nanobody phage display, scFv phage display, and Fab phage display.

Alpha Lifetech has a comprehensive M13/T7 phage display platform to ensure the construction and production of antibody libraries. We can also provide customized services, such as scFv phage display, Fab antibody production, and Fab antibody purification, to meet your needs.

Alpha Lifetech can provide a one-stop shop for your research needs, from phage display selection to NGS sequencing. In addition, Alpha Lifetech provides Fab antibody purification services.

Synthetic Fab Antibody Library Screening Service Process

Solid Phase Screening

After the phage Fab library was constructed, affinity screening was carried out, using the solid phase screening method.

One is to fix the antigen to the object's surface, and the antigen can be adsorbed on the activated surface through various non-covalent interactions. The antigen is diluted with a detergent buffer containing no interference adsorption and pH > 9 to increase the net charge of the protein molecule and promote hydrophilic interaction with the surface and then added to the drop plate for incubation with the phage Fab antibody library. The Fab binds to the solid phase antigen, so the Fab binding is stable. The unbound or unbound phage is then rinsed off, and the phage line is removed with a specific eluent. Repeat these steps several times. After 3 ~ 4 rounds of screening, Fab binding phage was prepared, the screened Fab binding antibody was identified, and the obtained positive clones were soluble. The specificity of Fab antibody was evaluated by Western Blot and immunohistochemistry. The affinity of Fab antibody was evaluated by BLI. After obtaining the target clone, the results were verified by NGS sequencing technology for Fab antibody purification.

However, this method has limitations: (1) Low molecular weight antigens, such as haptens or peptides, cannot be adsorbed on the surface alone and need to be coupled with carrier proteins; The structure of the antigen will change, and the antibody will not recognize the free antigen in the solution.

Another approach is to bind the target antigen to biotin, with fixed streptavidin used to capture the surface antigen. Proteins can be biotinized by side-chain chemical reactions, and haptens and short peptides can be synthesized biotinized. Before the addition of biotinylated antigens, the library needs to be pretreated and pre-incubated with streptavidin-coated microbeads to bind the streptavidin conjugate. The library was incubated with a biotinized antigen solution and magnetic beads were added. Although biotin can affect the physical and chemical properties of antigens, this method can adsorb antigens with conformational changes.

Fig.1 Biological screening of phage display antibody library. (Reference source: Antibody Phage Display - PubMed (nih.gov))

Whole-cell Phase Screening

Whole cells expressing surface antigens can be used as antigens for biological screening, In this method antibodies that bind to the target antigen can be obtained in a victorious environment. Because cells express a wide variety of surface molecules, only a small percentage will bind to the target molecule, so this method is difficult. Pretreatment is required before the start of the experiment to consume the overexpressed/non-target conjugate of the target antigen.

Cell Surface Screening

	Screening for Known Cell Surface Receptors	Screening for Unknown Cell Surface Receptors
Principle	In the culture dish, the blank cells that do not contain the target receptor in the antibody library are removed from the target antibody (that is, negative screening), and then the unadsorbed phage is combined with the target cell to obtain the antibody that binds to the target cell (that is, positive screening), and after several rounds of "negative screening - positive screening - amplification", the specific antibody can be enriched.	Screening for unknown receptors on the cell surface often results in antibodies that target a large number of molecular receptors on the cell surface, so cross-reactions between antibodies and normal tissue must be avoided. It is necessary to select suitable negative cells to screen the background and improve the signal-to-noise ratio.
Shortcoming	In the negative selection process, non-specific phages cannot be effectively shielded. In the positive screening process, some positive clones with small copy number are easy to be lost. Most of the enriched antibody clones are non-specific, and the number and diversity are far less than that of pure antigens.	In the complex antigen environment with uncertain receptor antigen properties and scarce functional receptor expression, screening is often interfered with receptor bias and diversity, resulting in low screening efficiency and high screening background. In addition, the technique is difficult to operate, the recovered phage is small, the antibody affinity is low, and the development value is not great.